GSM6892022: Blastocyst_WGBS_SCNT_rep1; Macaca fascicularis x Macaca mulatta; Bisulfite-Seq
ICSI and SCNT blastocysts were seeded into lysis buffer by mouth pipette and subjected to bisulfite conversion using the EZ DNA Methylation Direct Kit. Each replicates containing 10~11 blastocysts were collected for the library construction. A small amount of unmethylated Lambda DNA (Promega, D152A) was added to each sample before bisulfite conversion to serve as spike-in controls for evaluating bisulfite conversion efficiency. After column-based purification, DNA was complemented with the random primer Preamp and Klenow polymerase (Enzymatics, P7010-HC-L). This random priming was repeated five times in total. Second strands were synthesized using another random primer, Adapter 2. Final libraries were generated after PCR amplification with the Illumina universal PCR primer and Illumina indexed primer, and were then purified using SPRI beads. Final libraries were subjected to paired-end 150 bp sequencing on a NovaSeq 6000 sequencer (Illumina) with PhiX spike-in control.